Synthetic copolymers, such as those based on styrene and acrylate derivatives, have found widespread use as solid supports in a number of techniques of fundamental importance in modern biochemistry and related biomedical fields. Examples of such well-studied and refined methodologies include affinity chromatography, immobilized enzyme technology, and the solid-phase synthesis of nucleic acids and peptides. Most pertinent to the background of the present invention is the last of these examples, commonly known as solid-phase peptide synthesis (SPPS). The method of SPPS involves the stepwise construction from the C-terminus of a polymer-bound protected peptide, followed by its deprotection and release from the polymeric support to yield the free peptide, as illustrated in FIG. 1. The advantages of this approach are well documented and include the facile removal of reagents and by-products from the peptide intermediates at each stage of the synthesis, and the amenability of the method to automation.
First described by Merrifield in 1963, R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963), the method of SPPS represented a major advance in the field of peptide chemistry. Although many of the techniques and protocols associated with the method of SPPS have since been refined to such a degree that a vast number of peptides can be prepared on polymeric resins in routine fashion using sophisticated automated synthesizers, the Merrifield method does not lend itself well to some applications, such as the bulk synthesis of peptides on an industrial scale, and the condensation of peptide fragments. There is, therefore, an ongoing need for methods to complement Merrifield's SPPS approach.
One area of research that showed promise in this regard involved the use of polymeric reagents in peptide bond formation. This approach represented a fundamental departure from the Merrifield strategy in the sense that an insoluble polymeric support was utilized not simply as an anchor for the growing peptide but, in fact, participated in the coupling step as the activated carboxyl component. In this method, the protected peptide product is released into solution after coupling and can be separated from the polymeric resin by simple filtration. The first example of this approach was described by Fridkin et al. in 1966, M. Fridkin, A. Patchornik, E. Katchalski, J. Am. Chem. Soc. 88, 3164 (1966). Fridkin et al. examined the preparation of polymer-bound o-nitrophenyl active esters of N-blocked amino acids and the use of these reagents in the synthesis of a series of small protected peptides, as illustrated in FIG. 2. Since then, the application of such reagents to peptide synthesis has been investigated in a number of laboratories using a variety of polymeric active esters, as well as polymeric coupling agents. This method has, in some respects, emerged as a useful alternative to the Merrifield method. Among the advantages of the polymeric reagent approach cited by Patchornik et al., A. Patchornik, E. Nov, K. A. Jacobson, Y. Shai, "Polymeric Reagents and Catalysts," W. T. Ford, ed., ACS Symposium Series No. 308, are rapid, high-yield coupling reagents, peptide products of exceptional purity, and the option of recycling the used polymeric resin.
Within the realm of polymeric reagents applied to peptide synthesis, one area that has been virtually ignored, however, is the use of polymeric mixed anhydrides despite the fact that in solution-phase peptide chemistry amino acid activation through mixed anhydride formation represents a well-established method of peptide bond formation. J. Meienhofer, The Peptides, 1, 263-314 (Acedemic Press 1979).
The scientific literature contains several reports describing various types of polymer-bound mixed anhydrides that demonstrate the potential synthetic utility of such reagents. The synthesis and application of resin supported mixed anhydrides were described by G. E. Martin, et al., J. Org. Chem. 43, 4571 (1978), and S. Boivin, et al., Bull. Soc. Chim. France II, 201 (1984), but the materials of these reports lack the chemical stability required to make them viable general reagents. The synthesis and application of resin supported mixed carboxylic acids were described by M. B. Shambhu, et al., Tetrahedron Lett. 31, 143, (1973), and M. B. Shambhu, et al., J. Chem. Soc. Chem. Commun. 619, (1974), but the materials of these reports produce slow and incomplete reactions. The mixed anhydrides of these published reports are also unselective toward derivatizing reagents. Hence, high levels of unproductive reaction occur at the resin, which makes the resin difficult, if not impossible to recycle.
Activation of N-blocked .alpha.-amino acids via mixed anhydrides has also been known since 1950. See, T. Wieland et al., Ann. 569, 117 (1950). Unfortunately, the instability, both structural and chemical, of these intermediates forced workers to develop more useful alternatives, which typically have included: (1) coupling methods that activate the carboxyl group in situ, or (2) the formation of alternative activated intermediates such as acid chlorides, acid fluorides, active esters or mixed anhydrides of chloroformates.
The only known published example of the application of polymeric mixed anhydrides to peptide synthesis is the published work of V. K. Haridasan et al., J. Org. Chem. 52, 2662 (1987), who described the preparation of (o-NO.sub.2)Cbz-L-Phe-Gly-OEt using a polymeric dithiocarbonic-carboxylic acid mixed anhydride. The reagents of this method are mixed anhydrides of a carboxylic acid and a resin-supported dithiocarbamic acid. The Haridasan et al. method also requires the prior activation of the carboxylic acid to an acid chloride followed by formation of the mixed anhydride by reaction with the resin-supported dithiocarbamate salt. However, many carboxylic acids, including N-blocked amino acids, form unstable acid chlorides. Consequently, the common belief has been that resin-supported acid chlorides are not particularly useful as reagents. No known published examples of mixed anhydrides of N-blocked amino acids and resin-supported carboxylic acids have been reported, for example. The assumption has been that such materials would be unstable even if a synthetic route to them was discovered.